ES2720195T3 - A method of methylation detection - Google Patents

Abstract

A method of quantitatively screening for the methylation of a DNA region of interest in a biological sample, said method comprising: (i) contacting DNA from said biological sample with two or more methylation-sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplifying the digested DNA sample from step (i) by using one or more forward primers and one or more reverse primers, which primers target DNA sequences flanking one or more of the regions of the endonuclease methylation-specific restriction endonuclease recognition sequences of step (i); and (iii) quantifying the level of said methylated DNA region of interest, wherein said quantification does not require determining the ratio of the amplified DNA from step (ii) to a corresponding undigested sample.

Description

DESCRIPTION

A method of methylation detection

Field of the Invention

The present invention relates to a method of screening for the presence of methylated DNA in a biological sample. More particularly, the present invention relates to a quantitative screening method of the level of one or more methylated genes of interest without the need to use an undigested internal reference sample as a reference point against which the relative quantification is calculated. The present invention is useful in a variety of applications that include, but are not limited to, providing a simpler and more accurate means of determining the state of DNA methylation, such as in the context of the diagnosis or monitoring of conditions characterized by changes in the DNA methylation.

Background of the invention

The bibliographic details of the publications referred to by the author in this specification are collected in alphabetical order at the end of the description.

The reference in this specification to any previous publication (or information derived from it), or to any subject that is known, is not considered nor should it be considered as recognition or admission or any form of suggestion that that previous publication (or derived information of it) or known matter is part of the common general knowledge in the field of research referred to in this specification.

Colorectal cancer includes cancerous growths in the colon, rectum and appendix. With 655,000 deaths a year worldwide, it is the fourth most common form of cancer in the United States, and the third leading cause of cancer-related death in Western countries. Colorectal cancers arise from adenomatous polyps in the colon. These fungus growths are usually benign, but some become cancer over time. Localized colon cancer is usually diagnosed by a colonoscopy.

Invasive cancers that are confined within the wall of the colon (stages I and II of TNM) are curable with surgery. If left untreated, they spread to regional lymph nodes (stage III), in which up to 73% are curable by surgery and chemotherapy. Cancer that metastasizes in distant locations (stage IV) is usually not curable, although chemotherapy can prolong survival, and in rare cases, surgery and chemotherapy together have been cured to cure patients (Markowitz and Bertagnolli, 2009, N. Engl. J. Med. 361 (25): 2449-60). Radiation is used with rectal cancer.

Colorectal cancer is preceded by adenomas. Adenomas are benign tumors, or benign neoplasms, of epithelial origin that derive from glandular tissue or that exhibit clearly defined glandular structures. Some adenomas show recognizable tissue elements, such as a fibrous tissue (fibroadenomas) and an epithelial structure, while others, such as bronchial adenomas, produce active compounds that could lead to clinical syndromes.

Adenomas can progress to become an invasive neoplasm, and then they are called adenocarcinomas. Therefore, adenocarcinomas are defined as malignant epithelial tumors that arise from glandular structures, which are constituent parts of many organs of the body. The term adenocarcinoma also applies to tumors that show a glandular growth pattern. These tumors can be subclassified according to the substances they produce, for example mucous secreting adenocarcinomas and serous adenocarcinomas, or according to the microscopic arrangement of their cells in patterns, for example papillary and follicular adenocarcinomas. These carcinomas can be solid or cystic (cystadenocarcinomas). Each organ can produce tumors that show a variety of histological types, for example the ovary can produce mucinous carcinomas and cystadenocarcinomas.

The symptoms of colorectal cancer depend on the position of the tumor in the intestine, and if it has metastasized. Unfortunately, many of the symptoms can also occur in other diseases, and therefore the symptoms may not be conclusively diagnostic of colorectal cancer.

Local symptoms are more likely if the tumor is located closer to the anus. There may be a change in bowel habits (constipation or diarrhea of new occurrence in the absence of another cause), a feeling of incomplete defecation and a reduction in stool diameter. Tenesmus and the change in the shape of the stool are characteristic of rectal cancer. Low digestive hemorrhages, which include the passage of bright red blood in the stool, may indicate colorectal cancer, as well as the increased presence of mucus. The mane, black stool with a tarry appearance, usually occurs in upper gastrointestinal bleeding (such as due to a duodenal ulcer), but is sometimes found in colorectal cancer when the disease is located at the beginning of the large intestine.

Colorectal cancer spreads very frequently to the liver. This can happen inadvertently, but large deposits in the liver can cause jaundice and abdominal pain (due to the stretching of the capsule). If tumor metastasis obstructs the bile ducts, jaundice may be accompanied by other features of biliary obstruction, such as pale stools.

Colorectal cancer may take many years to develop, and early detection of colorectal cancer greatly improves the prognosis. Even modest efforts to implement colorectal cancer screening methods can result in a decrease in cancer deaths. Despite this, colorectal cancer screening rates remain low. There are currently several different tests for this purpose:

• Digital rectal exam: The doctor inserts a gloved and lubricated finger into the rectum to palpate for abnormal areas. Only tumors large enough to be palpated in the distal rectum are detected, but it is useful as an initial screening test.

• Analysis of fecal occult blood: an analysis in search of blood in the feces. Two types of tests can be used to detect fecal occult blood, that is, based on guaiac (chemical analysis) and immunochemical. The sensitivity of immunochemical analyzes is superior to that of chemical analyzes without an unacceptable reduction in specificity (Weitzel JN (December 1999). "Genetic cancer risk assessment. Putting it all together." Cancer 86 (11 Supl): 2483 -92).

• Endoscopy:

^o Sigmoidoscopy: An illuminated probe (sigmoidoscope) is inserted into the rectum and lower colon to check for polyps and other abnormalities.

^o Colonoscopy: An illuminated probe called a colonoscope is inserted into the rectum and the entire colon to look for polyps and other abnormalities that may be caused by cancer. Colonoscopy has the advantage that if polyps are found during the procedure, they can be removed immediately. You can also collect tissue for a biopsy.

• Double contrast barium enema (DCBE): First, a preparation is taken at night to clean the colon. An enema containing barium sulfate is administered, and then air is blown into the colon, which distends it. The result is a thin layer of barium on the internal surface of the colon that is visible on X-ray films. In this way a cancer or precancerous polyp can be detected. This technique may not detect the flat polyp (less common).

• Virtual colonoscopy replaces X-ray films of the double-contrast barium enema (mentioned above) with a scan using special computed tomography, and requires a special software for interpretation by the radiologist. This technique is approaching the sensitivity of colonoscopy for polyps. However, any polyp found should be removed by standard colonoscopy.

• Standard computed tomography is an x-ray method that can be used to determine the degree of spread of cancer, but it is not sensitive enough to be used for screening. Some cancers are found in CT scans performed for other reasons.

• Blood tests: The measurement of the patient's blood for elevated levels of certain proteins can provide an indication of the tumor burden. In particular, the high level of carcinoembryonic antigen (CEA) in the blood may indicate the metastasis of adenocarcinoma. Although these tests are often false positives or false negatives, and are not recommended for screening, they can be useful for studying disease recurrence. The biomarkers CA19-9 and CA 242 may indicate metastatic risks related to e-selectin, help in monitoring therapeutic progress, and assess disease recurrence. Recently an analysis has also appeared for the detection of methylated sequences of the Septin 9 gene in plasma to aid in the diagnosis of colorectal cancer.

• Positron emission tomography (PET) is a three-dimensional scanning technology in which a radioactive carbohydrate is injected into the patient, the carbohydrate accumulates in tissues with a high metabolic activity, and an image is formed by measuring the emission of radiation of carbohydrate Because cancer cells often have very high metabolic rates, it can be used to differentiate benign and malignant tumors. PET is not used for screening, and (still) does not have a place in routine diagnostic tests of colorectal cancer cases.

• Stool DNA analysis is an emerging technology in colorectal cancer screening. Premalignant adenomas and cancers release DNA markers from their cells that do not degrade during the digestive process, and remain stable in the stool. The capture, followed by PCR, amplifies the DNA to detectable levels for analysis.

• High levels of C-reactive protein as a risk marker.

Despite the existence of these tests, the diagnosis remains problematic. Most of the most sensitive tests are quite invasive and expensive, and therefore acceptance by patients is low. Therefore, the determination that changes in the methylation of certain genes is indicative of the development of neoplasms of the large intestine has been very significant, since it provides a highly sensitive and reliable means of screening for the onset of neoplasms of the large intestine.

There are a variety of methods that are currently available to identify altered methylation sites in cancer cells. The analysis of DNA methylation patterns and the distribution of 5-methylcytosine is usually carried out using a bisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA, 89: 1827-1831 , 1992). Specifically, DNA bisulfite treatment is used as a starting point for the analysis of methylation through the use of a variety of techniques. These include methylation-specific PCR (MSP) (Herman et al., Proc. Natl. Acad. Sci. USA, 93: 9821-9826, 1992) and restriction enzyme digestion of amplified PCR products from DNA converted with bisulfite (Sadri and Hornsby, Nucl. Acids Res. 24: 5058-5059, 1996; Xiong and Laird, Nucl. Acids Res. 25: 2532-2534, 1997). Treatment with sodium bisulfite converts all unmethylated cytosines of DNA into uracil by deamination, but leaves the residues of methylated cytosine intact. Subsequent PCR amplification replaces uracil residues with thymine and 5-methylcytosine residues with cytosines. The difference in the resulting sequences can be detected by the use of usual DNA sequence detection techniques, mainly by sequencing bisulfite treated DNA by specific methylation PCR. However, there are disadvantages to bisulfite-based methods, which includes the need for high initial concentrations of DNA due to the relatively strong action of sodium bisulfite. Another method of analyzing changes in methylation patterns is a PCR-based process that involves the digestion of native / natural DNA with restriction enzymes sensitive to methylation before PCR amplification (Singer-Sam et al., Nucleotide Acids Res. 18: 687, 1990; see also page 37 of document US2012 / 0264618). However, this method has a tendency to suffer a high degree of false positive signals (i.e., that there is methylation) due to incomplete digestion of unmethylated DNA, which is then mistakenly amplified in a subsequent PCR reaction. Although significant research has been carried out in an effort to improve this screening method, in particular to reduce the incidence of false positives resulting from incomplete digestion, this technology has not made it possible to eliminate the incidence of false positives. That is, although improvements have been developed that reduce the degree of incomplete digestion, a method that reliably and routinely completes complete digestion has not been developed. Therefore, to date, methods based on the use of methylation-sensitive restriction endonucleases have required PCR analysis of undigested and digested aliquots of the test sample, where the test sample is studied for this purpose. control, and the reading you get is really of a relative nature. Therefore, there is a current need to develop improved methods that do not suffer from these limitations.

In the work that led to the present invention, it has been determined that when the analysis related to methylation-sensitive restriction endonucleases sensitive to DNA methylation is based on the use of at least two methylation-sensitive enzymes together with digestion to 4-6 pg of DNA / endonuclease unit / hour, a level of digestion is achieved that is effectively complete, which means that amplification by PCR will not result in detectable amplicons due to the presence of undigested DNA. Furthermore, when amplification is carried out by quantitative PCR with the use of primers flanking the region of methylation-specific restriction endonuclease recognition sequences, absolute quantification is feasible, and the need to amplify a control sample is eliminated. internal digested to calculate relative quantitative values. This advance has significant implications from the point of view of utility in diagnosis, since it not only provides a simpler and cheaper method to analyze DNA methylation, but also a means to obtain an exact quantification with accuracy.

Summary of the invention

The scope of the present invention is determined by the appended claims 1 to 13.

Throughout this specification and the claims that appear below, unless the context requires otherwise, it will be understood that the word "understand", and variations such as "comprises" and "comprising", imply the inclusion of a mentioned entity or stage or group of entities or stages, but without the exclusion of any other entity or stage or group of entities or stages.

As used herein, the term "from" shall be deemed to indicate that a particular entity or group of entities has originated from the specified species, but has not necessarily been obtained directly from the specified source. In addition, as used herein, the singular forms "a,""one," and "the" include plural references, unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as usually understood by one skilled in the art to which this invention pertains.

The present specification contains nucleotide sequence information prepared through the use of the PatentIn Version 3.5 program, presented herein after bibliography. Each nucleotide sequence is identified in the sequence list by the numerical indicator <210>, followed by the sequence identifier (eg <210> 1, <210> 2, etc.). The length, type of sequence (DNA, etc.) and the organism of origin for each sequence are indicated by the information provided in the fields of the numerical indicators <211>, <212> and <213>, respectively. The nucleotide sequences mentioned in the specification are identified by the indicator SEQ ID N °: followed by the sequence identifier (eg SEQ ID N °: 1, SEQ ID N °: 2, etc.). The sequence identifier mentioned in the specification is correlated with the information provided in the field of the numerical indicator <400> in the sequence list, which is followed by the sequence identifier (eg <400> 1, <400 > 2, etc.). That is, SEQ ID N °: 1, as detailed in the specification, is correlated with the sequence indicated as <400> 1 in the sequence list.

In one aspect, a method of quantitative screening of the methylation of a region of DNA of interest in a biological sample is provided, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said region of methylated DNA of interest.

In another aspect, a method of quantitative screening of the methylation of a region of DNA of interest in a biological sample is provided, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample.

In another aspect, a method of quantitative screening of the methylation of a gene of interest in a biological sample is provided in particular, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said methylated gene region, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample.

In another aspect a method of quantitative methylation screening is provided in one or more of the BCAT1, IKZF1, IRF4, GRASP and CAHM gene loci in a biological sample, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more recognition sequences of methylation specific restriction endonucleases; Y

(iii) quantify the level of said or said methylated genes, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample.

In another aspect, a method of quantitative screening of the methylation of a region of DNA of interest in a biological sample is provided, and said method comprises:

(i) extract the DNA from said biological sample and establish the concentration of total DNA present in said sample;

(ii) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more recognition sequences of methylation specific restriction endonucleases; Y

(iv) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In another aspect, a quantitative screening method of methylation of a gene of interest in a biological sample is therefore provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA from said biological sample with two or more methylation sensitive restriction endonucleases, wherein one of said enzymes is a Type IIe enzyme, and digest said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample of step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of selected methylation-specific restriction endonucleases; Y

(iv) quantify the level of said methylated gene, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In a further aspect, therefore, a method of quantitative screening of the level of methylation of one or more of BCAT1, IKZF1, IRF4, GRASP and CAHM in a biological sample is provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA of said biological sample with HpaII and HhaI and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA regions flanking one or more regions of sequence sequences. recognition of selected HpaII and HhaI; and (iv) quantify the level of said methylated BCAT1, IKZF1, IRF4, GRASP and / or CAHM, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In a further aspect, a method of quantitative screening of the level of methylation of one or more of BCAT1, IKZF1, IRF4, GRASP and C ^to HM in a biological sample is provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA of said biological sample with HpaII, HhaI and ExoI and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA regions flanking one or more regions of sequence sequences. recognition of selected HpaII and HhaI; and (iv) quantify the level of said BCAT1, IKZF1, IRF4, GRASP and / or CAHM methylated, in which said quantification does not require determining the proportion of the amplified DNA of step (iv) with respect to a corresponding undigested sample.

In a further aspect, the method of the present invention is directed to the screening of methylated GRASP by the use of a digestion with HhaI / HpaII, and the amplification step is carried out by the use of:

(i) Direct primer: 5'-CAAGTTGAAGGTCCGAGAGC (SEQ ID N °: 2); Y

(ii) Reverse primer: 5'-CGCACTTCCTCAGAGTGAGA (SEQ ID N °: 3).

In another additional aspect, the method of the present invention is directed to the screening of methylated BCAT1 by the use of a digestion with HhaI / HpaII, and the amplification step is carried out by the use of:

(i) Direct primer: 5'-AGATCCCAAGGGTCGTAGC (SEQ ID N °: 4); Y

(ii) Reverse primer: 5'-ACTGCCCCAGGTCTTGCT (SEQ ID N °: 5).

In a further aspect, the method of the present invention is directed to the screening of methylated IKZF1 through the use of a digestion with HhaI / HpaII, and the amplification step is carried out by the use of:

(i) Direct primer: 5'-GGAGTTGCGGCTGAGAC (SEQ ID N °: 6); Y

(ii) Reverse primer: 5'-AGAGCGGGACACGGAGA (SEQ ID N °: 7).

Brief description of the drawings

Figure 1 is a graphical representation of methylation levels measured in the BCAT1 gene locus through the use of the method. Digestion with restriction enzymes was applied to test the state of methylation [DREAMS] to circulating DNA isolated from 4 mL of blood plasma. The concentration of the extracted DNA was determined by quantitative real-time PCR, and 3.6 ng of DNA was digested for 72 hours with HhaI and HpaII. The digestion mixture was then analyzed in quantitative PCR with DREAMS of BCAT1 (1.6 ng per duplicated PCR reaction). The value "60" was artificially assigned to the absence of a signal. The figure shows the mean values of the cycle threshold [Ct] measured in 1.6 ng of digested DNA isolated from blood plasma from patients confirmed by colonoscopy, which included 50 normal samples (clear circles) and 16 colorectal cancers (dotted circles: Stage 1, n = 1; yellow: Stage 2, n = 4; orange: Stage 3, n = 4; red: Stage 4, n = 4; blades: unknown stages of cancer, n = 3). POSCONT: positive control for methylation detection. 4 mL of plasma from healthy donors to which 5 ng of fully methylated DNA (Millipore) was added. NEGCONT: negative control for the detection of methylation. 4 mL of plasma from healthy donors. Samples were considered positive if a Ct signal was obtained. The DREAMS test of BCAT1 demonstrated a specificity of 96% and a sensitivity of 63%.

Figure 2 is a graphical representation of the methylation levels measured in the GRASP gene locus by using the method described in the legend of Figure 1. A total of 3.6 ng of plasma DNA extracted for 72 hours was digested with 72 hours. HhaI and HpaII. The digestion mixture was then analyzed in quantitative PCR with GRASP DREAMS (1.6 ng per duplicate PCR reaction). The value "55" was artificially assigned to the absence of a signal. The figure shows the mean values of the cycle threshold [Ct] measured in 1.6 ng of digested native DNA isolated from blood plasma from patients confirmed by colonoscopy, which included 50 normal samples (clear circles) and 16 colorectal cancers (dotted circles: Stage 1, n = 1; yellow: Stage 2, n = 4; orange: Stage 3, n = 4; red: Stage 4, n = 4; blades: unknown stages of cancer, n = 3). The samples were considered positive with Ct values <38. POSCONT: positive control for the detection of methylation. 4 mL of plasma from healthy donors to which 5 ng of fully methylated Dn (Millipore) was added. NEGCONT: negative control for the detection of methylation. 4 mL of plasma from healthy donors. The GRASP DREAMS trial demonstrated a specificity of 99% and a sensitivity of 69%.

Detailed description of the invention

The present invention is based, in part, on the determination that complete digestion by methylation-specific restriction endonucleases can be carried out effectively, in which the digestion is carried out with two or more methylation-specific restriction endonucleases. at 4-6 pg of DNA / endonuclease unit / hr. Furthermore, by designing the amplification stage to use primers flanking the regions of the restriction endonuclease recognition sequences, absolute quantification is feasible and the need to carry out the quantification in a relative manner against a sample of undigested internal control.

Therefore, in one aspect, a method of quantitative screening of the methylation of a region of DNA in a biological sample is provided, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said region of methylated DNA of interest.

More particularly, a method of quantitative screening of the methylation of a region of DNA of interest in a biological sample is provided, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample.

In one embodiment, step (ii) is carried out in the presence of one or more TaqMan probes.

The reference to a "DNA region of interest" should be understood as a reference to any region of DNA, whose methylation state is to be analyzed. This may be, for example, a gene, part of a gene, an intergenic region, a mitochondrial promoter or DNA. For this purpose, the reference to a "gene" should be understood as a reference to a DNA molecule that encodes a protein product, either a full-length protein or a protein fragment. It should be understood, however, that there are some genes or regions of genes, which have been identified, that are known to not necessarily produce a protein product. It should be understood, therefore, that the reference to a "gene" herein includes the reference to both types of genes. From the point of view of genomic DNA, the gene will generally be expected to include intronic and exonic regions. The region of DNA of interest can also be a non-coding portion of ^a genomic D ⁿ that is known to be not associated with any specific gene (such as the so-called "junk" DNA regions). The target region of DNA of interest can also be any region of genomic DNA produced by recombination, between 2 regions of genomic DNA or 1 region of genomic DNA and a region of exogenous DNA, such as a virus or an introduced sequence. It is not necessary that the DNA that is the object of the analysis is necessarily genomic DNA, although it is generally understood that the DNA produced by means of recombinant techniques, such as cDNA or DNA copies of non-coding RNAs, is not methylated. However, it should be understood that the present invention is extended to the analysis of any source of DNA that may be methylated.

Without limiting the present invention to any theory or mode of action, DNA methylation is universal in bacteria, plants and animals. DNA methylation is a type of chemical modification of DNA that is stable throughout cell division rounds, but does not imply changes in the organism's underlying DNA sequence. Chromatin and DNA modifications are two important features of epigenetics, and they play a role in the cell differentiation process, which allows cells to stably maintain different characteristics despite containing the same genomic material. In eukaryotic organisms, DNA methylation occurs only in carbon number 5 of the cytosine pyrimidine ring. In mammals, DNA methylation occurs mainly in carbon number 5 of the cytosine of a CpG dinucleotide. CpG dinucleotides constitute approximately 1% of the human genome.

70-80% of all CpGs are methylated. CpGs can be grouped into clusters called "CpG islands", which are present at the 5 'end of the regulatory regions of many genes, and are often unmethylated. In many pathological processes, such as cancer, gene promoters and / or CpG islands acquire abnormal hypermethylation, which is associated with inheritable transcriptional silencing. DNA methylation can influence gene transcription in two ways. First, DNA methylation can physically impede the binding of transcriptional proteins to the gene, thereby blocking transcription. Second, methylated DNA can bind to proteins known as proteins with methyl-CpG binding domain (MBDs). Then MBD proteins incorporate additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histones, so that a compact and inactive chromatin called silent chromatin is formed. This association between DNA methylation and chromatin structure is very important. In particular, the loss of methyl-CpG binding protein 2 (MeCP2) has been implicated in Rett syndrome, and protein 2 with methyl-CpG binding domain (MBD2) mediates in transcriptional silencing of hypermethylated genes in cancer

In humans, the DNA methylation process is carried out by three enzymes, DNA methyltransferases 1, 3a and 3b (DNMT1, DNMT3a, DNMT3b). DNMT3a and DNMT3b are believed to be de novo methyltransferases that establish DNA methylation patterns at the beginning of development. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to daughter chains during DNA replication. DNMT3L is a protein that is homologous to the other DNMT3s, but has no catalytic activity. Instead, DNMT3L helps de novo methyltransferases by increasing their ability to bind to DNA and stimulate its activity. Finally, DNMT2 has been identified as an "enigmatic" homolog of DNA methyltransferase, which contains the 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 cannot methylate DNA, but instead it has been shown to methylate a small RNA.

It should be understood, therefore, that the term "methylation" means the presence of a methyl group added by the action of a DNA methyltransferase enzyme to a cytosine base or bases in a region of a nucleic acid, eg DNA genomic

In one embodiment, said region of DNA of interest is a gene.

According to this embodiment, a method of quantitative screening of the methylation of a gene of interest in a biological sample is more particularly provided, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of sequence sequences. recognition of methylation specific restriction endonucleases; Y

(iii) quantify the level of said methylated gene region, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample.

In another embodiment, said gene is a mammalian gene.

In a further embodiment, said gene is a marker of neoplasia of the large intestine and, more particularly, BCAT1, IKZF1, CAHM, GRASP or IRF4.

These genes are specified herein by reference to both the name of the gene and a group of human chromosomal coordinates. Both the names of the genes and the chromosomal coordinates would be well known, and understood, by the person skilled in the art. The chromosomal coordinates for the regions tested in adjacent probe arrays of Nimblegen promoters correspond to the Hg18 version of the genome, while those describing the associated gene symbol correspond to the Hg19 version of the genome. In general, a gene can be identified routinely by its name, by means of which its sequence and chromosomal location can be obtained routinely, or by its chromosomal coordinates, by means of which it can also be obtained routinely The name of the gene and its sequence.

The reference to "genes" should be understood as a reference to all forms of these molecules and to fragments or variants thereof. As the person skilled in the art would appreciate, it is known that some genes exhibit allelic variation between individuals or single nucleotide polymorphisms. SNPs encompass insertions and deletions of varying size and repetitions of simple sequences, such as dinucleotide and trinucleotide repeats. Variants include nucleic acid sequences from the same region that share at least 90%, 95%, 98%, 99% sequence identity, that is, that have one or more deletions, additions, substitutions, inverted sequences, etc., with respect to the genes described herein. Therefore, it should be understood that the present invention is extended to such variants that, from the point of view of the present diagnostic applications, achieve the same result despite the fact that there may be minor genetic variations between acid sequences specific nuclei between individuals. Therefore, it should be understood that the present invention is extended to all forms of DNA that arise from any other mutation, polymorphic or allelic variation.

The chromosomal coordinates of Hg19 that correspond to the genes detailed above are the following: (1) BCAT: chr12: 24962958..25102393

(2) IKZF1: chr7: 50344378 ... 50472798

(3) IRF4: chr6: 391739..411443;

(4) GRASP: chr12: 52400748..52409671; Y

(5) CAHM: chr6: 163834097..163834982.

It should be understood that the reference to these genes includes 5 kb in position prior to the transcription start site of each of these genes.

As will be discussed in more detail below, the method of the present invention can be applied to the screening of the methylation of a gene, or the methylation of more than one gene can be adapted in a specific biological sample by means of different aliquots. DNA from the original biological sample, or in the context of a single aliquot that is amplified by the use of a multiplexed amplification method (for example, using fluorescently labeled probes, such as TaqMan hydrolysis probes or molecular beacons).

According to this embodiment, a method of quantitative methylation screening is provided in one or more of the BCAT1, IKZF1, IRF4, GRASP and CAHM gene loci in a biological sample, and said method comprises:

(i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more recognition sequences of methylation specific restriction endonucleases; Y

(iii) quantify the level of said or said methylated genes, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample. The DNA that is analyzed according to the method of the present invention is isolated from a biological sample. It should be understood that the reference to a "biological sample" is a reference to any sample of biological material derived from any source, such as an animal, plant or bacterial source, which includes, but is not limited to, cellular material, biofluids (e.g. blood or plasma), feces, tissue biopsy samples, surgical samples or a fluid that has been introduced into an animal's body and subsequently removed (such as, for example, the solution recovered from an enema). The biological sample that is analyzed according to the method of the present invention can be analyzed directly, or it may require some form of treatment before analysis. For example, a biopsy or surgical sample may require homogenization before analysis. Alternatively, a cell sample may require permeabilization before analysis. In addition, to the extent that the biological sample is not in liquid form, (if such a form is necessary for analysis), it may require the addition of a reagent, such as a buffer, to mobilize the sample.

To the extent that the region of DNA of interest is present in a biological sample, the biological sample can be analyzed directly, or if all or part of the nucleic acid present in the biological sample cannot be isolated before analysis. In another example, the sample can be partially purified, or enriched in another way before analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to enrich it in a subpopulation of particular interest. It is within the scope of the present invention to treat the target biological sample or molecules derived therefrom before analysis, for example, inactivating a live virus. It should also be understood that the biological sample may have been collected recently, or it may have been stored (for example, by freezing it) prior to the analyzes, or it may have been treated differently before the analyzes (such as by subjecting it to culture).

The choice of what type of sample is most suitable for analysis according to the method described herein will depend on the nature of the situation. To the extent that it is being screened with respect to the onset or predisposition at the beginning of a large intestine neoplasm, for example, said sample is preferably a stool sample (feces), enema, surgical removal, tissue biopsy or sample of blood (eg whole blood, serum, leukocyte layer or plasma)

More preferably, said biological sample is a blood sample, biopsy sample or stool sample. As detailed herein, the method of the present invention is based on the determination that a quantitative reading of DNA methylation can be performed, in which a complete digestion of DNA can be performed by restriction endonucleases sensitive to methylation, followed by amplification by using primers designed to flank the recognition sequence of methylation-specific restriction endonucleases. This determination makes it possible for the skilled person to dispense with the analysis of an undigested internal sample against which the results of the analysis are analyzed, obtained through the use of prior art methods. These prior art methods provide only a relative reading with respect to methylation levels. However, because the method of the present invention enables complete digestion to be carried out, the application of a quantitative amplification method that is based on the use of amplification primers directed at the DNA flanking the recognition sequence of methylation-specific restriction endonucleases enable absolute quantification. It should be understood that the reference to a "complete" digestion, in the context of the method of the present invention, is a reference to a level of digestion efficiency that results in no detectable PCR signal of the amplification of the DNA that has not been completely digested. Therefore, the skilled person would appreciate that there may be some degree of incomplete digestion, but this will be so insignificant that this incompletely digested DNA will not result in a detectable PCR signal when using the PCR method of the present invention. In this situation, it is therefore considered that the digestion of DNA is effectively complete. In addition, this improvement Significant efficacy of the digestion stage has enabled the analysis of very low amounts of DNA, such as up to 1 ng. From the point of view of collecting enough biological material for the analyzes, this is very significant.

For this purpose, in one embodiment, the DNA is preferably extracted from the biological sample by any suitable extraction method, and the concentration of total DNA present in the sample is established. Methods for carrying it out would be well known to the person skilled in the art, and include, for example, phenol / chloroform extraction, cesium chloride gradients, CHELEX or silica column, or microsphere methods. By virtue of the extraction and establishment of the concentration of the DNA present in the biological sample of interest, the ability to carry out the method of the present invention with very low initial concentrations of DNA is facilitated.

Therefore, in one embodiment, a method of quantitative screening of the methylation of a region of DNA in a biological sample is provided, and said method comprises:

(i) extract the DNA from said biological sample and establish the concentration of total DNA present in said sample;

(ii) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more recognition sequences of methylation specific restriction endonucleases; Y

(iv) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In one embodiment, said region of DNA of interest is a gene.

In another embodiment, 1-20 ng of the DNA extracted in step (i) is subjected to the digestion of step (ii). In another embodiment, 1-15 ng or 1-10 ng of DNA is used. More particularly, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng or 9 ng of DNA can be used.

Once the sample DNA is ready for analysis, it is digested with two or more methylation sensitive restriction endonucleases. It should be understood that the reference to a "methylation sensitive restriction endonuclease" is a reference to a restriction endonuclease, whose DNA cleavage activity is blocked or hindered when a particular basis of its DNA recognition sequence is methylated. Therefore, unmethylated DNA is cleaved, and therefore is unable to be amplified by primers designed to amplify by crossing the endonuclease cleavage site. Only methylated DNA will remain intact, and thereby generate an amplification product.

As the skilled person would appreciate, this method, therefore, depends on the selection and use of enzymes for which the relevant recognition sequence is present in the region of the DNA of interest. Because methylation-specific restriction endonucleases are well known and well characterized, the selection of an appropriate endonuclease to enable analysis of the gene of interest is within the skill of the person skilled in the art. For example, in Table 1, below, an exemplary list of methylation-specific restriction endonucleases is detailed together with the DNA recognition sequence relevant to each enzyme.

TABLE 1

From the point of view of the selection of the enzyme mixture for use in any specific situation, in addition to selecting enzymes for which the recognition sequences are present in the gene of interest, it is especially useful if the recognition sequences of two or more enzymes are located from proximally within a subregion of the gene of interest. This is useful from the point of view of amplification, since it minimizes the length of the DNA between the outermost 5 'and 3' recognition sequences, and thereby minimizes the size of the intact DNA fragment that needs to be present in the sample for the method to work. This enables a sensitive analysis of a highly fragmented DNA, such as that found in the circulating acellular DNA. As detailed herein, because methylation-specific restriction endonucleases are well known and well characterized, in particular in the context of their recognition sequences, the selection of a suitable mixture of two or more enzymes for the use with a specific gene of interest is within the experience of the person skilled in the art.

For this purpose, it should be understood that the reference to the primers of interest that target DNA regions "flanking" the "region of methylation-specific restriction endonuclease recognition sequences" is a reference to the use of one or more direct and inverse primers that are located proximally at 5 'and 3', respectively, with respect to the subregion of the target DNA comprising the recognition sequences. "Proximally" means that it is not necessary that the hybridization sites of the primers be immediately adjacent to the outermost recognition sequences, but are located to enable amplification of the region of recognition sequences without unnecessarily amplifying large segments of DNA that are outside this region. In this regard, the direct primers can be located in a position before the cutting site of the relevant endonucleases, and the reverse primers in a position after the cutting site of the relevant endonucleases, so that they amplify by crossing the cutting site, or it can hybridize across the cutting site, so that they will hybridize only to DNA that has not been cut.

It should be appreciated that when the two or more enzymes are ischysmerers, and therefore cut in the same recognition sequence, the relevant amplification region will comprise only one recognition sequence, and the direct and inverse primers will therefore be designed. to hybridize to DNA sequences that flank this region of recognition sequences. When methylation-specific restriction endonucleases that cut into different recognition sequences are used, it should be understood that the "region of methylation-specific restriction endonuclease recognition sequences" of interest refers to the subregion of the gene within which they are grouped recognition sequences. In this regard, in one embodiment, the two or more methylation-specific restriction endonucleases are selected such that if they are not isoschemomers, they are, however, specific to recognition sequences that are located proximally with each other to minimize size. of the amplicon that would be generated when there is methylation.

The primers of interest are said to "flank" this region of methylation-specific restriction endonuclease recognition sequences when they are designed to hybridize to DNA sequences that are located 5 'and 3' from the outer ends of the sequence pool. of recognition of interest, or that hybridize crossing the external 5 'and 3' cutting sites. Although it would be appreciated that one or more of the methylation-specific restriction endonucleases can cut into a variety of positions throughout the entire gene, the skilled person may choose to amplify the entire gene, in which case the "selected" region of sequences of recognition of methylation-specific restriction endonucleases is indeed the complete gene. Alternatively, the skilled person can choose a specific subregion into which one or more recognition sequences are grouped, and amplify only this minor region (and not any other recognition sequence present in other parts of the gene). In this case, this smaller subregion is the "selected" region of methylation-specific restriction endonuclease recognition sequences.

As detailed hereinbefore, the method of the present invention is based on the determination that a combination of two or more of said methylation-specific restriction endonucleases, together with a digestion at 4-6 pg of said DNA by unit of said endonucleases per hour, achieves a complete digestion of DNA in analysis. This finding is especially significant when it is considered that it had previously been taught that not all methylation sensitive restriction endonucleases are suitable for use in this type of application. In particular, it was understood that Type IIe enzymes, such as HpaII, created problems since they could not carry out complete digestion. Therefore, an undigested DNA background should be expected (Levenson and Melnikov, Expert Rev. Molecule. Diagn. 11 (8): 807-812, 2011). The method of the present invention, however, enables the use of any restriction endonuclease sensitive to methylation.

In one embodiment of the present invention, the two or more methylation-specific restriction endonucleases include at least one Type IIe enzyme, more particularly HpaII. According to this embodiment, therefore, a method of quantitative screening of the methylation of a gene of interest in a biological sample is provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA from said biological sample with two or more methylation sensitive restriction endonucleases, wherein one of said enzymes is a Type IIe enzyme, and digest said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample from step (ii) by using one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more regions of selected methylation-specific restriction endonuclease recognition sequences; Y

(iv) quantify the level of said methylated gene, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In another embodiment, said Type IIe enzyme is HpaII.

In another embodiment, the digestion step is carried out by using the HpaII and HhaI methylation-specific restriction endonucleases together with the single stranded DNA exonuclease, ExoI.

In another embodiment, the digestion step is carried out by using the methylation-specific restriction endonucleases HpaII, HhaI and ExoI.

According to these embodiments, in a further embodiment said gene is BCAT1, IKZF1, IRF4, GRASP or CAHM.

According to this embodiment, therefore, a method of quantitative screening of the level of methylation of one or more of BCAT1, IKZF1, IRF4, GRASP and CAHM in a biological sample is provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA of said biological sample with HpaII and HhaI and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA regions flanking one or more regions of sequence sequences. recognition of selected HpaII and HhaI; and (iv) quantify the level of said methylated BCAT1, IKZF1, IRF4, GRASP and / or CAHM, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

In another embodiment a method of quantitative screening of the level of methylation of one or more of BCAT1, IKZF1, IRF4, ^g R ^a S ^p and CAHM in a biological sample is provided, and said method comprises:

(i) establish the concentration of total DNA present in said sample;

(ii) contacting 1-10 ng of DNA of said biological sample with HpaII, HhaI and ExoI and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour;

(iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA regions flanking one or more regions of sequence sequences. recognition of selected HpaII and HhaI; and (iv) quantify the level of said methylated BCAT1, IKZF1, IRF4, GRASP and / or CAHM, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample.

It should be understood that the reference to a "methylation-specific restriction endonuclease" is a reference to all forms of this enzyme, which includes any isoform arising from the alternative splicing of the mRNA of the enzyme of interest, or variants allelic or polymorphic.

It is necessary that digestion of DNA with methylation-specific restriction endonucleases develops over a period of time that is based on the ratio of 4-6 pg of said DNA per unit of said endonucleases per hour. It should be understood that the reference to the "unit" of said endonucleases is a reference to the total units of activity of the endonuclease mixture that has been selected for use. The unit of activity of each endonuclease is defined by the manufacturer as the amount of enzyme needed to digest a specified amount of a model DNA substrate (e.g., 1 pg of lambda phage DNA) at a specified time (e.g. ., 1 hr) at a specified temperature (eg, 37 ° C) in a specified buffer. The total units in a particular mixture are defined as the sum of the units of the individual component endonucleases of the mixture. Therefore, because the initial amount of DNA is known, together with the number of endonuclease units to be used, the proper digestion time can be calculated. For example, if 18 ng of DNA will be digested by using 56 units of a mixture of endonucleases, the digestion would have to develop for at least 72 hours to carry out a complete digestion within the meaning of the present invention. In another example, when it is desired to digest 5 ng of DNA with an enzyme mixture of 110 units, a digestion time of at least 8 hours would be used.

The digestion of interest can be carried out at any suitable temperature, such as at 37 ° C. As the skilled person would understand, different restriction enzymes can function optimally at different temperatures. The optimum temperature at which to use a specific enzyme is well known in the art.

It should be understood that, in addition to digesting the DNA with the specific methylation restriction endonucleases of interest, additional reagents may also be included as deemed useful. For example, one can also choose to use reagents that additionally help control digestion, such as double oligonucleotide molecules containing recognition sites, self-complementary oligonucleotides or an oligonucleotide stimulator such as 5-TATAGCCGGCTATA (SEQ ID N °: 1). These mechanisms help digestion control are well known to those skilled in the art. In one embodiment, when HpalI is used, an oligonucleotide HpalI stimulator is also introduced into the digestion. In another example, an exonuclease that digests single stranded DNA, such as 0.15 U of exonuclease I per hour, can also be used. In addition, suitable internal controls may be included, such as a control confirming that complete digestion has been achieved.

As detailed herein, one of the important advances of this method is the fact that complete digestion of the DNA sample can be obtained routinely and reliably. The prior art methods, to date, have been limited by the fact that complete digestion of a DNA sample has not been reliably feasible. For this reason, all prior art methods have required that an analysis DNA sample be divided into two aliquots, in which an aliquot is digested and the corresponding sample has not. Both samples are then amplified, and the results of the amplification are expressed as a proportion of the results of the amplification of the digested sample with respect to the results of the amplification of the undigested sample. Therefore, these results have been of a relative nature. The prior art methods did not allow absolute quantification. It should be understood that this undigested control sample of the prior art is referred to in step (iv) of the present method as a "corresponding undigested sample". This sample, therefore, is undigested, but, nevertheless, it is amplified by using the same primers and amplification conditions specified in step (iii) for the digested sample. It should also be understood that this sample is also called an "undigested internal reference sample" in the literature.

It should be understood that the reference to "contacting" a sample with a methylation-specific restriction endonuclease is a reference to facilitate the mixing of methylation-specific restriction endonucleases with the DNA sample so that interaction can occur, and therefore digestion. The means to accomplish this objective would be well known to those skilled in the art.

The digested DNA sample is quantitatively amplified by the use of primers flanking the region of the methylation-specific restriction endonuclease recognition sequences. As detailed herein, this "region" can be selected to cover all or a substantial part of the length of the gene, in which case the amplicons generated will be quite long. Alternatively, the region may correspond to a much shorter stretch of the gene, in which one or more recognition sequences are grouped. In this case, the amplicons that were generated would be significantly shorter.

It should be appreciated that, when carrying out the amplification step, this can be done by using any of several suitable techniques. For example, when more than one pair of direct / reverse primers is used, aimed at selecting two or more regions of different recognition sequences as a target, all these primers can be introduced into a single sample and amplify the sample by using a multiplexed amplification technique. Alternatively, it is possible to choose to divide the digested sample from step (ii) into more than one aliquot, in which each aliquot is amplified by using a different pair of primers. It should also be understood that the skilled person can choose to adapt this method to use multiple groups of primers, aimed at amplifying only one region of methylation-specific restriction endonuclease recognition sequences, but in which the multiple primers reflect the application of a nested PCR reaction.

It should be understood that the reference to a "primer" is a reference to any molecule comprising a nucleotide sequence, or functional derivatives or analogs thereof, whose function includes both hybridization to a DNA sequence flanking the region of methylation-specific restriction endonuclease recognition sequences such as amplification of the 5 'DNA sequence of that region. It should be understood that the primer may comprise components other than nucleic acid. For example, the primer may also comprise a distinct nucleic acid tag, such as a fluorescent or enzymatic tag, or some other component other than nucleic acid that facilitates the use of the molecule as a probe, or that facilitates its detection . In another example, the primer may be a protein nucleic acid comprising a peptide skeleton that exhibits nucleic acid side chains. Preferably, said primer is a single stranded DNA oligonucleotide.

It should be understood that the reference to a "direct primer" is a reference to a primer that amplifies the target DNA of the DNA sample of interest by hybridizing with the reverse strand of the target DNA.

It should be understood that the reference to a "reverse primer" is a reference to a primer that amplifies the DNA target of the DNA sample of interest and in the PCR hybridizing with the direct strand of the target DNA.

The design and synthesis of primers suitable for use in the present invention would be well known to those skilled in the art. In one embodiment, the primer of interest has a length of 4 to 60 nucleotides, in another embodiment it has a length of 10 to 50 nucleotides, in another embodiment it has a length of 15 to 45 nucleotides, and in another embodiment it has a length of 20 at 40 nucleotides.

From the point of view of the number of primers used in the method of the invention, the person skilled in the art can determine it. With respect to the total number of primers, the variables that require consideration are the size and number of regions of the DNA to be amplified and the distance between the sequences with which the primers hybridize. To amplify PCR fragments that are larger than about 1 kb, primers can be designed to work in a nested PCR method and hybridize at intervals of approximately 500 bases.

As detailed herein, when multiple regions of DNA are to be amplified, the skilled person can design multiplexed amplification reactions. Alternatively, several individual amplification reactions can be carried out that each use a single pair of primers. These methods become relevant when two or more regions of different methylation-specific restriction endonuclease recognition sequences are being amplified, or when the methylation of more than one gene is to be analyzed. In this case, after digestion of the sample with the methylation-specific restriction endonucleases, the sample can be divided into two aliquots, if one wishes to analyze two genes (such as BCAT1 and IKZF1), and then each aliquot is amplified by the use of the group or groups of direct and inverse primers directed to the regions of relevant methylation-specific restriction endonuclease recognition sequences of that gene. Alternatively, a multiplexed reaction can be carried out with a single sample in which the reaction is multiplexed from the point of view of the use of primers directed to the region of methylation-specific restriction endonuclease recognition sequences selected from a gene, and the use of another group of primers targeting the region of methylation-specific restriction endonuclease recognition sequences selected from the other gene. As the skilled person would know, multiplexed reactions can be designed to be carried out with two, three or more groups of primers in the context of two or more regions of methylation-specific restriction endonuclease recognition sequences and / or two or more genes . It should be understood that the proper design of multiplexed or nested amplification reactions is within the skill of the person skilled in the art.

In one embodiment, to the extent that the method of the present invention is directed to methylated GRASP screening through the use of a digestion with HhaI / HpaII, the amplification step is carried out by using: (i) Primer Direct: 5'-CAAGTTGAAGGTCCGAGAGC (SEQ ID N °: 2); Y

(ii) Reverse primer: 5'-CGCACTTCCTCAGAGTGAGA (SEQ ID N °: 3).

In another embodiment, to the extent that the method of the present invention is directed to methylated BCAT1 screening through the use of a digestion with HhaI / HpaII, the amplification step is carried out by using: (i) Primer Direct: 5'-AGATCCCAAGGGTCGTAGC (SEQ ID N °: 4); Y

(ii) Reverse primer: 5'-ACTGCCCCAGGTCTTGCT (SEQ ID N °: 5).

In another embodiment, to the extent that the method of the present invention is directed to the screening of methylated IKZF1 through the use of a digestion with HhaI / HpaII, the amplification step is carried out by the use of: (i) Primer Direct: 5'-GGAGTTGCGGCTGAGAC (SEQ ID N °: 6); Y

(ii) Reverse primer: 5'-AGAGCGGGACACGGAGA (SEQ ID N °: 7).

It should be understood that the reference to "quantitatively amplify" the digested DNA is a reference to carry out any suitable method of quantitative amplification of the DNA. This includes, for example, quantitative PCR or quantitative linear amplification. In one embodiment, real-time quantitative forms of PCR are used, such as, for example, TaqMan (Holland et al., Proc. Natl. Acad. Sci. USA, 88: 7276-7280, 1991; Lee et al., Nucleic Acid Res. 21: 3761-3766, 1993), Beacon or Scorpion. Quantitative amplification methods are described, eg, in pat. from the USA No. 6,180,349; 6,033,854; and 5,972,602, as well as, eg, in Gibson et al., Genome Research 6: 995-1001 (1996); DeGraves, et al., Biotechniques 34 (1): 106-10, 112-5 (2003); Deiman B, et al., Mol. Biotechnol 20 (2): 163-79 (2002). These methods are well known to the person skilled in the art. Without limiting this embodiment to any theory or mode of action, the qPCR is carried out in the presence of three oligonucleotides, a direct and inverse primer flanking the region to be amplified and a probe that hybridizes between the two primers. It can also be carried out in the presence of two oligonucleotides and a fluorescent intercalating dye, such as SYBR Green. In the context of the above method, the probe is doubly labeled with a 5 'fluorescent indicator and a 3' damper (or vice versa). When the probe is intact, the dye quencher absorbs the fluorescence of the indicator due to its proximity. After hybridization to the PCR product, the probe is cleaved by the 5 'to 3' exonuclease activity, for example, from Taq DNA polymerase. This cleavage releases the indicator of the damper, and thus an increased fluorescence signal is obtained that can be used to estimate the initial methylation level of the mold. Alternatively, instead of using a labeled probe that requires cleavage, a probe is used, such as, for example, a molecular beacon (see, for example, Mhlanga and Malmberg, Methods 25: 463-471, 2001). Molecular beacons are single-stranded nucleic acid molecules with a stem and loop structure. The structure of the loop is complementary to a region between the primers. The structure of the stem is formed by hybridization of two "arms" complementary to each other, which are on each side of the probe (loop). A fluorescent moiety is attached to one arm and a quencher moiety is attached to the other arm, which inhibits any detectable fluorescence when the molecular beacon is not bound to an objective sequence. After the binding of the region of the loop to its target nucleic acid, the arms are separated and the fluorescence is detectable.

As detailed herein, one of the significant advantages of the present method is that it enables absolute quantification by using methods such as the threshold value of the cycle of a real-time PCR instrument, in which the presence of Any signal indicates the presence of methylated DNA. This can be accomplished by comparing with a standard curve performed under the same conditions of qPCR, which contains known amounts of natural DNA digested with non-restriction endonucleases, or completely methylated DNA digested with restriction endonucleases. Therefore, the use of internal controls is not necessary to compensate for incomplete digestion, and the reading obtained by the method of the invention therefore provides an absolute value instead of a relative proportion. However, it should be understood that one may wish to compare the quantitative reading obtained by the present method with a quantitative result of a normal individual or of a previous sample obtained from the patient in question. This would allow monitoring of changes in DNA methylation levels.

The facilitation of the interaction of the primer with the target DNA can be carried out by any suitable method. Those skilled in the art will know those methods. For this purpose, it should be understood that the primers can be incorporated into the reaction tube at any suitable time. Although incorporation generally occurs before the start of the initial amplification cycles, the incorporation of one or more additional primers can be carried out after the initial amplification cycles. The mode of incorporation of the primers will depend on how the skilled person wishes to carry out the amplification reaction but, in general, for simplicity of use and to avoid contamination, it is normally desirable to be able to carry out the entire reaction in a single tube. . However, any other method can be used to perform the steps of the invention.

Although the preferred application of this method is to study the levels of methylation in order to diagnose the onset of a disease (such as the development of a neoplasia or the predisposition to it), the detection of inverse changes in the levels of said methylation in certain circumstances, for example, to monitor the efficacy of a therapeutic or prophylactic treatment aimed at modulating a neoplastic condition, such as the development of an adenoma or adenocarcinoma. For example, when elevated levels of methylation indicate that an individual has developed a condition characterized by the development of an adenoma or adenocarcinoma, screening for a decrease in methylation levels after the start of a therapeutic treatment regimen can be achieved. use to indicate an effective removal of neoplastic cells. In another example, this method can be used to analyze the tissue of the margins of a tumor removal to determine if the entire margin of the tumor has been removed.

The present method can therefore be used in the diagnosis, prognosis, classification, prediction of the risk of a disease, the detection of the recurrence of a disease, the selection of the treatment of various types of neoplasms and the monitoring of neoplasms Cancer can be detected at any stage of progression, such as primary, metastatic, and recurrent cancers. In addition, this method has applications in any other context in which DNA methylation analysis is necessary.

By using the development of a neoplasm as a non-limiting example, the present invention provides methods to determine if a mammal (eg, a human being) has a neoplasm, if a biological sample obtained from a mammal contains neoplastic cells or DNA derived from neoplastic cells, estimate the risk or probability that a mammal develops a neoplasm, monitor the efficacy of an antineoplastic treatment, or select the appropriate antineoplastic treatment in a mammal with cancer. Such methods are based on the determination that many neoplastic cells have a state of methylation different from that of normal cells. Therefore, by determining whether or not a cell contains differentially methylated sequences, it is possible to determine that a cell is neoplastic.

The method of the invention can be used to study individuals who are known or suspected to have a malignancy, or as a routine clinical analysis, that is, in an individual in whom it is not necessarily suspected that they have a malignancy. Additional diagnostic tests may be carried out to confirm the state of the neoplasm in the individual.

In addition, the present methods can be used to study the effectiveness of a course of treatment. For example, it You can study the efficacy of an antineoplastic treatment by monitoring DNA methylation over time in a mammal that has cancer. For example, the reduction or absence of methylation in any of the relevant diagnostic sequences of a biological sample obtained from a mammal after a treatment, compared to a level in a sample obtained from the mammal before, or sooner in, the treatment , indicates an effective treatment.

The method of the present invention, therefore, is useful as a timely analysis or as continuous monitoring of those individuals who are considered to be at risk of developing a disease, or as monitoring the efficacy of therapeutic or prophylactic treatment regimens. In these situations, the location of the modulation of the methylation levels in one or more kinds of biological samples is a valuable indicator of the status of an individual or the efficacy of a therapeutic or prophylactic regimen that is being used at that time. Therefore, it should be understood that the method of the present invention is extended to monitoring the increases or decreases of the levels of methylation in an individual with respect to their normal level, or with respect to one or more specific levels of prior methylation from a biological sample of said individual. The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1

Digestion with restriction enzymes to test the state of methylation in the BCAT1 and GRASP gene loci The protocol includes reagents and methods to extract free circulating DNA from blood plasma and digest free circulating DNA with HhaI methylation sensitive enzymes, HpaII and Exonuclease I. Then the level of methylation of BCAT1 and GRASP is determined.

1. "Isolation of circulating nucleic acids in serum / plasma" by QIAGEN:

to. QIAGEN® 50 mini columns

b. Tube Extenders (20 ml) 2 X 25

c. VacConnectors 50

d. ACL buffer 2 x 85 mL

and. ACB buffer 2 x 190 mL

F. ACW1 buffer 2 x 20 mL

g. ACW2 buffer 2 x 25 mL

h. Ethanol for buffer extraction 2 x 25 mL

i. AVE buffer (purple caps) 4 x 1984L

j. QIAGEN Proteinase K 4 x 7 mL

k. Carrier RNA (1 g / L) 2 x 160L

2. Control plasma samples

to. POSCONT: added 5 ng of fully methylated DNA 2 x 4 mL

b. NEGCONT: mixed plasma of> 8 patients without disease 2 x 4 mL

Equipment Needed

Extraction of DNA

The procedure described herein is recommended to process a batch of 24 samples that includes 22 samples (4.0 mL of centrifuged plasma twice) plus a negative and a positive control ("POSCONT 'and" NEGCONT "). The resulting natural DNA is sufficient for QC analysis (if necessary) and subsequent tests of restriction digestion

Extraction Procedure

System

4. Put on a new disposable lab coat. Discard the lab coat at the end of the day.

6. Put on a pair of gloves and make sure that the gloves extend over the cuffs of the lab coat. New gloves should be worn when contamination is suspected; and after each incubation and before handling clean components (eg columns, Eppendorf tubes, 96-well plates).

7. When working outside the biosafety cabinet with open containers, put on a hair net. 8. Preheat the water bath to 60 ° C.

9. Thaw a positive control plasma tube, a negative control plasma tube and 22 samples at room temperature for 30-60 minutes in a biosafety cabinet. Ensure that the samples are properly separated in the cabin to allow complete defrosting.

10. While the plasma is thawing:

to. Place a vial of carrier RNA (1 g / L) at room temperature. Once thawed, vortex for 5 seconds and centrifuge in a desktop microcentrifuge for 5 seconds.

b. Add 148 L of thawed carrier RNA (1 g / L) to the ACL Buffer (85 mL) and mix by inverting 10 times.

c. Label 24 x 50 mL tubes (lid and side) with corresponding unique numbers specific to each sample for extraction (eg 1-24).

Enzymatic digestion

11. Once the samples are thawed, begin extraction for each sample by transferring the plasma to a 50 mL tube pre-labeled for reagent addition. The following components (which include the sample) should be added in order to all tubes before continuing with the next component. Use a new tip for each stage:

to. 4 mL of plasma, to all tubes, then:

b. 400 L of Proteinase K from QIAGEN, to all tubes and shake, and then finally:

c. 3.2 mL of ACL Buffer (containing carrier RNA) to all tubes.

12. Close the lid of the first 50 mL tube and invert the tube once. Repeat for the remaining 23 tubes. 13. Thoroughly mix in a vortex shaker each tube for exactly 30 sec. (Vortexing should be carried out in groups of two tubes).

14. Incubate the plasma-extraction solutions in the water bath at 60 ° C for 30 min.

15. Prepare the QIAvac 24 plus collector in a biosafety cabinet as described by the manufacturer.

Lisis

16. Remove the 50 mL tubes from the water bath.

17. Centrifuge each of the 50 mL tubes in a centrifuge for 10 seconds to ensure that all lysate is at the bottom of the tube.

18. Place the tubes in a biosafety cabinet and add 7.2 mL of ACB Buffer to each tube by using a new tip for each sample.

19. Close the lid of the first tube and invert once. Repeat for the remaining 23 tubes. Mix thoroughly by pulsatile vortexing for 20 seconds at a time. Incubate the lysate-ACB buffer mixture on ice for 5 minutes. (The procedure should be carried out in groups of two tubes at once). Column purification

20. Carefully decant the ACB buffer-lysate mixture into the tube extender of the corresponding numbered QIAamp mini column.

21. Turn on the vacuum pump (pump pressure must reach 1000 mbar).

22. During the filtration of the sample prepare two QIAcubes for DNA extraction as described by the manufacturer.

QIAcube extraction

23. Once the samples have been completely removed through the columns, turn off the vacuum pump and release the pressure at 0 mbar. Carefully remove and discard the tube extender.

24. Place the mini spin columns on the rotor adapters previously prepared. Distribute the 24 samples equally in each of the two QIAcubes (12 samples each).

25. Open the caps of the reagent bottles of the QIAcubes and make sure that all the components are in the correct position.

26. Operate the QIAcube as specified by the manufacturer. Ensure that the QIAcube completes the pre-check.

27. Clean the QIAvac manifold as described by the manufacturer

Second manual elution for increased performance

28. After completion of the procedure, remove the rotor adapters from the QIAcube and place them on a rack of the QIAcube. Transfer the elution tube-column assembly to a suitable rack.

29. For each sample, reapply the eluate (~ 37 L) in the center of the membrane.

30. Close the column lid and incubate at room temperature for 3 minutes.

31. Centrifuge the sample tubes at 20,000 g for 1 min at room temperature to elute the natural DNA.

32. Discard the columns and transfer 36 L of each natural DNA sample to a 96-well PCR plate.

Methylation Sensitive Digestion

Digestion Method

Determination of DNA concentration

35. Add 10 L of water without nucleases to 24 wells of a 96-well PCR plate.

36. Transfer 2.5 L of each of the 24 DNA samples extracted to a well containing 10 pL water without nucleases.

37. Place caps in strips on each of the wells

38. Analyze 2x5 pL of the 1/5 dilution prepared in step 36 in the CFF1 assay described below.

Include a standard curve of serial dilutions to 1/4 of fully methylated DNA (Millipore) to allow quantification of the concentration of the extracted natural DNA.

CFF1 test:

CFF1 direct primer: TAA GAG TAA TAA TGG ATG GAT GAT G (SEQ ID N °: 8)

CFF1 reverse primer: CCT CCC ATC TCC CTT CC (SEQ ID N °: 9)

CFF1 [HEX] Probe ATG GAT GAA GAA AGA AAG GAT GAG T [BHQ1] (SEQ ID N °: 10) Reaction of 15 | jl containing a final concentration of 630 nM of Primers, 200 nM of Probe and 3 mM of MgCl ² in 1X Platinum Taq buffer.

PCR cycle conditions: 1x Cycle at 95 ° C for 2 min, followed by 50x cycles at 95 ° C for 10 sec, 60 ° C for 50 sec.

39. Use CFF1 data to calculate the concentration per µl of the original plasma DNA.

40. Dilute each of the 22 clinical samples of DNA, POSCONT and NEGCONT to 0.36 ng / jL (25 jL of each required sample).

Enzymatic digestion of extracted DNA

41. For each gene locus (ie, GRASP or BCAT1): Prepare a master digestion mixture by using the following volumes (sufficient for 22 samples, POSCONT, NEGCONT, 7 standards and a blank control:

Do not vortex enzymes: Mix all reagents thoroughly before use.

42. Pipette 35 µl of the master digestion mixture prepared in step 41 into 32 wells of a 96-well PCR plate as follows:

43. Add 10 µl of the diluted DNA samples prepared in Step 40 to the appropriate wells and prepare a CpG DNA standard starting at 2500 pg and add it to column 7 as indicated above (add 10 µl of water to the White).

44. Seal the plate by using BioRad sealing film and centrifuge briefly.

45. Mix the plate in a MixMate device for 5 minutes at 1200 RPM, briefly centrifuge and mix again in the MixMate device for another 5 minutes at 1200 RPM.

46. Centrifuge briefly and transfer the plate to an Axygen thermal cycler. Incubate at 37 ° C for 72 hrs with the lid heated to 60 ° C (adjust the volume to 45 pl).

Methylated DNA Detection

PCR settings and data interpretation

47. 1 hour before the planned end of digestion a PCR master mix is prepared as follows (sufficient for 70 wells):

BCAT1:

BCAT1 HpaN_HhaI

No. of wells

70

PCR conditions:

PCR conditions:

48. Transfer the PCR master mix to an 8-well strip and pipette 60 pl of master mix into columns 1-8 of a 96-well LC480 plate.

49. Once the digestion has developed for 72 hrs, adjust the heated cap of the AXYGEN thermocycler to 110 ° C and place the plate for 10 minutes at 98 ° C to inactivate HpaII / HhaII and ExoI. 50. Remove the plate from the device and centrifuge briefly to bring all the liquid to the bottom of the well.

51. Carefully remove the sealing film and, using a 100 pl multichannel pipette, transfer 20 pl (in duplicate) from the digestion reaction to the appropriate well of the qPCR plate (see below)

52. Seal the LC480 plate with sealing film, briefly centrifuge and mix in a MixMate for 30 seconds at 1100 RPM. Centrifuge again to collect the liquid from the bottom of the wells.

53. Transfer the LC480 board to a thermal cycler and operate it by using the following settings:

95 ° C for 2 mins, followed by 55x cycles at 95 ° C for 15 secs, 60 ° C for 30 secs and 72 ° C for 45 secs (Acquisition on the FAM channel).

54. Export Ct values and concentrations for each well for analysis.

Table 1: Primers / Probes

EXAMPLE 2

DREAMS IKZF1 test

The DNA was extracted and quantified to allow the generation of 25 pL of natural DNA at 0.36 ng / pL as described in Example 1. A total of 10 pL (3.6 ng of DNA) was digested in one volume Total digestion of 45 pL as described in Example 1. The state of methylation in the IKZF1 gene locus was analyzed in an 80 pL PCR reaction with an initial amount of 20 pL of digested DNA as described below. :

Test:

Liberty Taq 1x master mix (Life Technologies), 3 mM MgCl ² (final), 200 nM direct primer, 200 nM reverse primer, 100 nM probe.

PCR cycles:

EXAMPLE 3

DREAMS double test of IKZF1 / BCAT1

The DNA was extracted and quantified to allow the generation of 25 pL of natural DNA at 0.36 ng / pL as described in Example 1. A total of 10 pL (3.6 ng of DNA) was digested in one volume Total digestion of 45 pL as described in Example 1. The state of methylation in the gene loci, BCAT1 and IKZF1, was simultaneously analyzed in an 80 pL PCR reaction with an initial amount of 20 pL of digested DNA as outlined below:

Test:

Liberty Taq 1x master mix (Life Technologies), 3 mM MgCl ² (final), 200 nM direct primer, 200 nM reverse primer, 100 nM probe.

PCR cycles:

Bibliography

DeGraves, et al., Biotechniques 34 (1): 106-10, 112-5 (2003)

Deiman B, et al, Mol. Biotechnol 20 (2): 163-79 (2002)

Frommer et al, Proc. Natl Acad. Sci. USA, 89: 1827-1831, 1992 Gibson et al., Genome Research 6: 995-1001 (1996)

Herman et al, Proc. Natl Acad. Sci. USA, 93: 9821-9826, 1992 Holland et al, Proc. Natl Acad. Sci. USA, 88: 7276-7280, 1991

Lee et al., Nucleic Acid Res. 21: 3761-3766, 1993

Levenson and Melnikov, Expert Rev. Molecule. Diag. 11 (8): 807-812, 2011 Markowitz and Bertagnolli (2009). N. Engl. J. Med. 361 (25): 2449-60 Sadri and Hornsby, Nucl. Acids Res. 24: 5058-5059, 1996

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Xiong and Laird, Nucl. Acids Res. 25: 2532-2534, 1997

Document US2012 / 0264618 A1 (Oct. 18, 2012)

Claims (13)

1. A method of quantitative screening of the methylation of a region of the DNA of interest in a biological sample, and said method comprises: (i) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (ii) quantitatively amplify the digested DNA sample from step (i) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more of the regions of the methylation-specific restriction endonuclease recognition sequences of the endonucleases of step (i); Y (iii) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (ii) with respect to a corresponding undigested sample. 2. The method according to claim 1, wherein step (ii) is carried out in the presence of one or more probes, such as TaqMan probes. 3. The method according to any of claims 1 to 2, wherein said DNA is a gene, such as a mammalian gene. 4. The method according to claim 3, wherein said mammalian gene is a marker of neoplasia of the large intestine. 5. The method according to claim 4, wherein said gene is one or more of the BCAT1, IKZF1, IRF4, GRASP and CAHM gene loci. 6. The method according to any one of claims 1 to 5, wherein said method comprises: (i) extract the DNA from said biological sample and establish the concentration of total DNA present in said sample; (ii) contacting DNA of said biological sample with two or more methylation sensitive restriction endonucleases and digesting said DNA at 4-6 pg of said DNA per unit of said endonucleases per hour; (iii) quantitatively amplify the digested DNA sample from step (ii) through the use of one or more direct primers and one or more reverse primers, whose primers are directed to the DNA sequences flanking one or more of the sequences of recognition of methylation-specific restriction endonucleases of the endonucleases of step (ii); Y (iv) quantify the level of said region of methylated DNA of interest, in which said quantification does not require determining the proportion of the amplified DNA of step (iii) with respect to a corresponding undigested sample. 7. The method according to claim 6, wherein 1-20 ng of the DNA extracted in step (i) is subjected to the digestion stage of (ii). 8. The method according to claim 7, wherein 1-15 ng or 1-10 ng of DNA is used. 9. The method according to claim 8, wherein 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng or 9 ng of DNA are used. 10. The method according to any one of claims 1 to 9, wherein said two or more methylation-specific restriction endonucleases include at least one Type IIe enzyme, such as HpaII 11. The method according to claim 10, wherein the digestion step is carried out by using the methylation-specific restriction endonucleases HpaII, HhaI and ExoI exonuclease. 12. The method according to claim 10, wherein said method is directed to the screening of: (a) Methylated GRASP through the use of digestion with HhaI / HpaII, and the amplification step is carried out through the use of: (i) Direct primer: 5'-CAAGTTGAAGGTCCGAGAGC (SEQ ID N °: 2); Y (ii) Reverse primer: 5'-CGCACTTCCTCAGAGTGAGA (SEQ ID N °: 3); or (b) Methylated BCAT1 through the use of digestion with Hhal / HpalI, and the amplification step is carried out through the use of: (i) Direct primer: 5'-AGATCCCAAGGGTCGTAGC (SEQ ID N °: 4); Y (ii) Reverse primer: 5'-ACTGCCCCAGGTCTTGCT (SEQ ID N °: 5); or (c) IKZF1 methylated by the use of digestion with Hhal / HpalI, and the amplification step is carried out by the use of: (i) Direct primer: 5'-GGAGTTGCGGCTGAGAC (SEQ ID N °: 6); Y (ii) Reverse primer: 5'-AGAGCGGGACACGGAGA (SEQ ID N °: 7). 13. The method according to any of claims 1 to 12, wherein said biological sample is a blood sample, biopsy sample or stool sample.